Nitrogen-efficient monocot plants

ABSTRACT

Methods of increasing nitrogen utilization efficiency in monocot plants through genetic modification to increase the levels of alanine aminotransferase expression and plants produced there from are described. In particular, methods for increasing the biomass and yield of transgenic monocot plants grown under nitrogen limiting conditions compared to non-transgenic plants are described. In this way, monocot plants may be produced that maintain a desired yield while reducing the need for high levels of nitrogen application.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.11/644,321, filed Dec. 21, 2006, now U.S. Pat. No. 8,288,611, whichclaims the benefit of U.S. Application No. 60/753,818, filed Dec. 23,2005, the contents of which are hereby incorporated by reference in thepresent disclosure in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:595792001110SeqListing.txt, date recorded: Dec. 11, 2012, size: 73 KB).

FIELD OF INVENTION

The invention relates to monocot plants having enhanced nitrogenutilization efficiency (NUE), to methods for enhancing NUE in monocotplants, and to methods of increasing biomass and seed yield in monocotplants grown under nitrogen limiting conditions. This invention alsorelates to monocot antiquitin promoters.

BACKGROUND OF THE INVENTION

In many ecosystems, both natural and agricultural, the productivity ofplants is limited by the three primary nutrients: nitrogen, phosphorousand potassium. The most important of these three limiting nutrients isusually nitrogen. Nitrogen sources are often the major components infertilizers (Hageman and Lambert, I. Corn and Corn Improvement, 3rd ed.,Sprague & Dudley, American Society of Agronomy, pp. 431-461, 1988).Since nitrogen is usually the rate-limiting element in plant growth,most field crops have a fundamental dependence on inorganic nitrogenousfertilizer. The nitrogen source in fertilizer is usually ammoniumnitrate, potassium nitrate, or urea.

Each year, approximately 85 to 90 million metric tons (MMt) ofnitrogenous fertilizers are added to the soil worldwide. This is up fromonly 1.3 MMt in 1930 and from 10.2 MMt in 1960. It is predicted toincrease to 240 MMt by the year 2050 (Tilman et al., Proc. Nat. Acad.Sci. USA. 96: 5995-6000, 1999). It is estimated that 50% to 70% of theapplied nitrogen is lost from the plant-soil system. Because NO3- issoluble and not retained by the soil matrix, excess NO3- may leach intothe water and be depleted by microorganisms. In fact, most of theapplied nitrogen is rapidly depleted by soil microorganisms, leaching,and other factors, rather than being taken up by the plants.

Increased nitrogen utilization efficiency by plants would have a numberof beneficial effects. For example, nitrogen utilization efficientplants would be able to grow and yield better than conventional plantsin nitrogen poor soils. The use of nitrogen efficient plants wouldreduce the requirement for the addition of nitrogenous fertilizers tocrops. Since fertilizer application accounts for a significantpercentage of the costs associated with crop production, such areduction in fertilizer use would result in a direct monetary savings.

A reduction in fertilizer application would also lessen theenvironmental damage resulting from extensive nitrogenous fertilizeruse. These detrimental effects of nitrogenous fertilizer use on theenvironment are manifested in increased eutrophication, acid rain, soilacidification, and the greenhouse effect.

Monocots represent a large percentage of the crops grown on the world's3.7 billion acres of cultivable land. In the United States alone, over80 million acres of maize, 59 million acres of wheat, 4 million acres ofbarley and 3 million acres of rice were planted in 2004.

Given the worldwide requirements for monocots and the diminishingfertility of existing fields, it is desirable to generate monocot plantsthat are able to grow under suboptimal nutrient conditions. One meansfor accomplishing this goal is to generate monocot plants that canutilize nitrogen more efficiently. Such monocot plants would have theadvantage of being able to grow in soils that are poorer in nitrogen, asa result of being able to more efficiently use the nitrogen that isavailable. Additionally, such monocot plants may demonstrate enhancedproductivity in soils that have normal nitrogen levels as well.

Rice is routinely used as the model crop for genetic and physiologicalstudies in other monocot crops including maize, wheat, sugarcane,barley, sorghum, rye and grass. Because of its importance as a modelcrop, rice was the first crop plant to be sequenced. The InternationalRice Genome Sequencing Project, a consortium of publicly fundedlaboratories, completed the sequencing of the rice genome in December2004. Rice has a small, diploid genome that is well conserved andsyntenic across monocots. It is easily transformed and transgenicstudies have been performed in rice to study a number of phenotypictraits, including flowering, abiotic stress response, diseaseresistance, drought tolerance, and morphological development.

Because of the critical importance of nitrogen to plant growth, previousstudies have attempted to increase the efficiency of nitrogenutilization in plants using a variety of means. These methods haveincluded conventional breeding programs directed toward the developmentof plants that are more efficient at nitrogen utilization. Recombinantdeoxyribonucleic acid (DNA) and transgenic plant methods have also beenemployed in an attempt to generate nitrogen efficient plants.

A variety of different genes have been over expressed in dicot plants toincrease nitrogen use efficiency with variable results (for review, seeGood et al., Trends Plant Sci 9:597-605, 2004). However, monocots anddicots differ from each other in many ways including morphologically,developmentally, metabolically, phenotypically, and genetically. Becauseof these numerous differences, it would not be predictable thatsuccessful whether successful approaches to increase nitrogenutilization efficiency in dicots would necessarily work in monocots.

In the dicot canola, over expression of the enzyme alanineaminotransferase (AlaAT) under the direction of the Brassica turgorgene-26 (also known as antiquitin) promoter elevates AlaAT levels andincreases NUE (U.S. Pat. No. 6,084,153). However, whether overexpression of AlaAT would increase NUE in monocot plants has not beenpreviously reported.

Increasing NUE within monocot plants is desired within the art.

SUMMARY OF THE INVENTION

The invention addresses the need for monocot plants with enhanced growthcharacteristics and nitrogen utilization efficiencies when grown underlow nitrogen conditions by providing such plants and methods forgenerating transgenic monocot plants with elevated levels of AlaAT.

In one aspect, the invention provides transgenic monocot plantsincluding a recombinant DNA sequence encoding an AlaAT. The transgenicmonocot plant may be barley, rice, sugar cane, maize, sorghum, rye,wheat, or grass. Grass includes lawn, turfgrass, forage and the like.Preferably, the AlaAT is operably linked to a promoter, most preferably,a monocot antiquitin promoter. Seeds from the transgenic monocot plantsare also provided.

In other embodiments, transgenic rice, maize, wheat, sorghum, barley,and sugar cane include a recombinant DNA sequence encoding an AlaAT andseeds therefrom.

In another aspect of the invention, a method of producing a transgenicmonocot plant is provided including the steps of: (1) selecting anucleic acid encoding an AlaAT, (2) selecting a promoter that isoperable in a monocot plant, (3) coupling the selected nucleic acid tothe selected promoter to form a genetic construct, (4) transforming amonocot plant cell with the genetic construct to form a transformedcell, and (5) growing a transgenic monocot plant from the transformedcell to produce a transgenic plant. In this embodiment, overexpressionof AlaAT causes at least a 5% to 7.5%, 7.5 to 10%, 10 to 15% or 15 to20%, or more increase in plant biomass and/or seed yield when expressedin a transgenic monocot plant compared to the plant biomass or seedyield of a comparable monocot plant not expressing this construct whenthe plants are grown under suboptimal nitrogen conditions.

In other embodiments of the invention, a similar methods of producingtransgenic rice, maize, wheat, and sorghum plants are provided.

In yet another aspect of the invention, transgenic monocot plants aredescribed wherein the transgenic monocot plant expresses a recombinantAlaAT and exhibits at least a 5% increase in plant biomass or seed yieldcompared to biomass or seed yield of a comparable plant lacking therecombinant AlaAT. Also described are seeds produced from the transgenicmonocots. The monocots include but are not limited to, maize, wheat,rice, barley and rye.

A method for increasing biomass of a monocot plant by contacting andintroducing into a plant an AlaAT coding region in operative linkagewith monocot antiquitin promoter is described. Similar methods forincreasing seed yield of a plant and are also provided.

The nucleic acids encoding AlaAT that are used in the genetic constructsof these inventions may be derived from any organism preferably a plant,and most preferably from a monocot plant including, but not limited to,barley, rice, sugar cane, rye, wheat, maize, or grass.

In yet another aspect, the invention provides an isolated monocotantiquitin promoter sequence. The monocot promoter sequence may be frombarley, rice, sugar cane, maize, sorghum, rye, wheat, or grass. Incertain embodiments, it is a sorghum promoter that includes SEQ ID NO: 9or an active fragment thereof. In other embodiments, it is a maizepromoter that includes SEQ ID NO: 10 or an active fragment thereof.

Also provided are methods of directing expression of a target gene bycontacting and introducing into a plant a target gene in operativelinkage with a monocot antiquitin promoter.

Also described are genetic constructs, transformed plants, and plantseeds including a monocot antiquitin promoter sequence operativelylinked with a target gene. Preferably, the target gene encodes anitrogen utilization protein, such as, for example, a high affinitynitrate transporter, a low affinity nitrate transporter, an ammoniumtransporter, an ammonia transporter, an amino acid transporter, alaninedehydrogenase, glutamine synthetase, asparagine synthetase, glutamatesynthase, glutamate 2:oxogluturate amino transferase, asparaginase,glutamate dehydrogenase, nitrate reductase, aspartate aminotransferase,or AlaAT.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of the key steps in nitrogenutilization in a plant cell. Nitrate (NO₃ ⁻) is transported into theplant cell and converted to nitrite (NO₂ ⁻) by nitrate reductase (NR).Nitrite is translocated from the cytoplasm to the chloroplast where itis reduced by nitrite reductase (NiR) to ammonium (NH₄ ⁺). Glutaminesynthetase (GS) functions in assimilating or recycling ammonium. Anenzyme couple glutamine synthetase (GS)/glutamate synthase (GOGAT)catalyzes the conversion of glutamine (Gln) to glutamate (Glu).Glutamate is a building block of many amino acids. In addition, alanineis synthesized by the enzyme AlaAT from pyruvate and glutamate in areversible reaction.

FIG. 2 parts A-D show an alignment of the amino acid sequences (SEQ IDNO:s 29 to 45) of AlaAT from various organisms. Note that some ofsequences used for these alignments are truncated sequences whichcontain less than the complete sequence of the cited AlaAT. Thealignment was performed using the methionine (M) of the barley AlaATsequence as the reference first residue.

FIG. 3 parts A-C show an alignment of the amino acid sequences (SEQ IDNO:s 29 to 40) of AlaAT from various plant species. Note that some ofsequences used for these alignments are truncated sequences that containless than the complete sequence of the cited AlaAT. The alignment wasperformed using the methionine (M) of the barley AlaAT sequence as thereference first residue.

FIG. 4 shows the nucleotide sequence for the OSAnt1 promoter of theinvention (SEQ ID NO:1). The sequence was isolated using a blastn searchof the National Center for Biotechnology Information (NCBI) databaseusing the nucleotide sequence (366-3175 bp) of the Brassica btg26 gene(Stroeher et al., 1995, Plant Mol. Biol. 27:541-551) to identify thehomologous rice nucleotide sequence (accession number AF323586). Thissequence was then used in turn against the TIGR Oryza sativa sequencingproject (see: tigr.org/tdb/e2k1/osa1/), as set out in Example 1. Theputative TATA box is shown in bold and the primers used in PCRamplifying the sequence from the rice genome are underlined.

FIG. 5 shows a schematic representation of the steps for producing thegenetic construct OsAnt1pro-Gus, using the reporter genebeta-glucuronidase (GUS) in accordance with the method described inExample 1.

FIG. 6 shows a schematic representation of the steps for producing thegenetic construct OsAnt1pro-AlaAT in accordance with the methoddescribed in Example 1.

FIG. 7 shows expression of the GUS reporter gene directed by the OsAnt1promoter of the invention. Expression is present in the cell expansionarea of root tips of developing roots (panel A); in root hairs ofdeveloping roots (panel B); and in lateral roots of roots (panel C) ofan Oryza sativa plant transformed with the genetic constructOsAnt1pro-Gus as shown in FIG. 5, in accordance with the methoddescribed in Example 1. Darkly stained areas indicate expression of theGUS reporter gene.

FIG. 8 shows the average dry weight biomass (grams) of Oryza sativaplants transformed with the genetic construct OsAnt1pro-AlaAT as shownin FIG. 6 compared to the average dry weight biomass (grams) of control,wild-type Oryza sativa plants grown under the same growth conditions asgiven in Example 1.

FIG. 9 shows the average total seed weight (grams) of seeds collectedfrom Oryza sativa plants transformed with the genetic constructOsAnt1pro-AlaAT as shown in FIG. 6 compared to the average total seedweight (grams) of seeds collected from control, wild-type Oryza sativaplants grown under the same growth conditions as given in Example 1.

FIG. 10 shows the relationship between dry weight biomass (grams) andtotal seed weight (grams) for each transgenic plant.

FIG. 11 shows the nucleotide sequence of the sorghum antiquitin promoterof the invention (SEQ ID NO:9). The sequence was derived from accessionCW033386 as described in Example 5 and includes 443 nucleotides ofsequence upstream of the ATG start codon of a sorghum antiquitin gene.

FIG. 12 shows the nucleotide sequence of a partial maize antiquitinpromoter (SEQ ID NO:10). The sequence was derived from accessionBH215004 as described in Example 5 and contains 204-bp upstream of amaize antiquitin gene.

DETAILED DESCRIPTION

Monocot plants having enhanced NUE, methods for enhancing NUE in monocotplants, and methods of increasing biomass and seed yield in monocotplants grown under nitrogen limiting conditions are described herein.Limiting nitrogen conditions are conditions under which the plantbiomass or seed yield are reduced as a result of reduced nitrogenlevels. Under such conditions, the plant biomass or seed yield can beincreased by increasing the amount of available nitrogen byfertilization or other means. Limiting conditions are also known assuboptimal conditions.

Nitrogen assimilation and metabolism in plants occurs through thecoordinated action of a variety of enzymes acting upon a variety ofsubstrates (FIG. 1). Nitrogen assimilation occurs primarily through theactivities of glutamine synthetase (GS) and glutamate synthase (GOGAT).From the GS-GOGAT cycle, glutamate is used as a nitrogen source tosupply nitrogen for other required metabolic reactions. The metabolicflow of nitrogen is principally mediated by transamination reactions inwhich an amino group of glutamate is transferred to other carbonskeletons. The transfer of the amino group from glutamate to these othercarbon skeletons results in the disposition of nitrogen in more readilyusable forms such as other amino acids like aspartate or alanine.Examples of such enzymes are the aminotransferases. FIG. 1 shows thereaction catalyzed by the enzyme AlaAT which catalyzes the transfer ofan amino group from glutamate to pyruvate thus generating alanine.

While not limiting the invention to a particular mechanism, it isbelieved that over expression of AlaAT increases nitrogen efficiency bydepleting the available pools of nitrogen storing amino acids such asglutamate, which in turn leads to upregulation of the uptake andassimilation pathways in the plant. By transferring an amino group fromglutamate to pyruvate, the action of AlaAT depletes the pools ofglutamate, a nitrogen storage compound. Moreover, the pool ofalpha-ketoglutarate is replenished. To compensate for glutamatedepletion, the plant increases uptake and assimilation of nitrogen torestore the balance. The increased uptake and assimilation activityallows the plant to more effectively utilize lower (suboptimal) levelsof nitrogen present in the soil.

Monocot antiquitin promoters, such as rice, sorghum, and maize, are alsodescribed herein for use with any type of coding regions of interest.

DEFINITIONS

The language “transgenic” refers to a monocot plant that contains anexogenous nucleic acid molecule that can be derived from the samemonocot plant species, from a heterologous plant species, or from anon-plant species.

A “promoter” is a regulatory nucleic acid sequence, typically locatedupstream (5′) of a gene or protein coding sequence that, in conjunctionwith various cellular proteins, is responsible for regulating theexpression of the gene or protein coding sequence. Such promoters can bethe full length promoter or active fragments thereof. By “activefragment” is meant a fragment that has at least about 0.1%, preferablyat least about 10%, and more preferably at least about 25% of theactivity of a reference promoter sequence as tested via methods known tothose of skill in the art for detecting promoter activity, e.g.,measurement of GUS reporter gene levels. DNA sequences necessary foractivity can be identified by synthesizing various fragments and testingfor expression or introducing point mutations in certain regions andtesting for loss of activity.

Heterologous fragments of promoters or other promoter sequences may becombined to mediate the activity of a promoter sequence. For example,the CaMV 35S promoter or other known promoter sequences may be combinedwith the promoter sequence described herein to mediate expression of acoding region of interest.

The language “coding region of interest” or “target gene” includes anygene that is desirably expressed in one or more than one plant tissue.Likewise, a “target protein” refers to any protein that is desirablyexpressed in one or more than one plant tissue. Examples of a codingregion of interest which may advantageously be utilized in conjunctionwith the methods described herein include nucleic acid sequences thatencode one or more than one protein involved in nitrogen assimilation,nitrogen utilization, nitrogen uptake or a combination thereof.

The term “elevated levels” of a protein of interest, as used herein inreference to protein levels in a transgenic monocot plant, means higherlevels of protein as compared to the protein levels of a correspondingmonocot plant variety lacking the transgene such as an over expressednucleic acid molecule encoding an AlaAT.

The gene constructs described herein can also include further enhancers,either translation or transcription enhancers, as may be required. Theseenhancer regions are well known to persons skilled in the art and caninclude the ATG initiation codon and adjacent sequences. The initiationcodon must be in phase with the reading frame of the coding sequence toensure translation of the entire sequence. The translation controlsignals and initiation codons can be from a variety of origins, bothnatural and synthetic. Translational initiation regions may be providedfrom the source of the transcriptional initiation region or from thestructural gene. The sequence can also be derived from the promoterselected to express the gene and can be specifically modified toincrease translation of the messenger ribonucleic acid (mRNA).

The gene constructs of the invention can further include a3′-untranslated (or terminator) region that contains a polyadenylationsignal and other regulatory signals capable of effecting mRNA processingor gene expression. Nonlimiting examples of suitable 3′-regions are the3′-transcribed non-translated regions containing a polyadenylationsignal of Agrobacterium tumor-inducing (Ti) plasmid genes such as thenopaline synthase (Nos gene), plant genes such as the soybean storageprotein genes, and the small subunit of the ribulose-1,5-bisphosphatecarboxylase (ssRUBISCO) gene.

By “operatively linked” or “operative linkage” it is meant that theparticular sequences interact either directly or indirectly to carry outan intended function, such as mediation or modulation of geneexpression. The interaction of operatively linked sequences may bemediated, for example, by proteins that interact with the operativelylinked sequences.

The term “exogenous” as used herein in reference to a nucleic acidmolecule means a nucleic acid molecule originating from outside theplant. An exogenous nucleic acid molecule can have a naturally occurringor non-naturally occurring nucleotide sequence. One skilled in the artunderstands that an exogenous nucleic acid molecule can be aheterologous nucleic acid molecule derived from the same plant speciesor a different plant species than the plant into which the nucleic acidmolecule is introduced. Alternatively, it can be a nucleic acid moleculederived from a non-plant species such as fungi, yeast, bacteria or othernon-plant organisms.

The following description is of a preferred embodiment.

Overview of Alanine Aminotransferases (AlaATs)

As a general class of enzymes, aminotransferases are pyridoxalphosphate-dependent enzymes that catalyze reactions known astransamination reactions. The transamination reaction catalyzed byaminotransferases involves the transfer of an α-amino group from anamino acid to the α-keto position of an α-keto acid. In the process, theamino acid becomes an α-keto acid while the α-keto acid acceptor becomesan α-amino acid. The specific aminotransferase, AlaAT, utilizesglutamate as the amino group donor and pyruvate as the amino groupacceptor. Transamination of pyruvate to form alanine is found invirtually all organisms. Accordingly, enzymes with AlaAT activity arealso found in virtually all organisms as well. This group of AlaATsforms a basis for the isolation and selection of the AlaATs of theinvention.

Identification of AlaATs

Because most organisms possess AlaAT activity and enzymes, a number ofmethods can be used to identify and isolate these sequences fromdifferent species. Given the strong correlation between structure andfunction, one may use knowledge of the sequences of known members of theAlaAT family to collect additional family members that can serve ascandidate AlaATs for use in the invention.

Database searching: One method that can be used to generate a group ofAlaAT sequences for use in the invention is database searching. Becausethe genomes of a number of organisms have been sequenced, computer-baseddatabase searching based on amino acid or nucleic acid homology willreveal sequences which are homologous to a known AlaAT that is used asthe query sequence. One common tool for such computer database searchingis the BLAST program available from the NCBI. The NCBI Basic LocalAlignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.215(3):403-410, 1990) is available from several sources, including theNational Center for Biotechnology Information (NCBI, Bethesda, Md.) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. It can be accessedat the NCBI website. A description of how to determine sequence identityusing this program is available at the NCBI website. An example of usinga BLAST program to identify members of the AlaAT family is described inExample 7. The use of computer programs such as Softberry and PSORT canbe used to determine the subcellular localization of these enzymes toexclude enzymes that are targeted to less optimal sites, i.e., to theperoxisome.

Among the methods for sequence alignment which are well known in the artare the programs and alignment algorithms described in: Smith andWaterman, J. Mol. Biol. 147(1):195-197, 1981; Needleman and Wunsch, J.Mol. Biol. 48(3):443-453, 1970; Pearson and Lipman, Proc. Natl. Acad.Sci. U.S.A. 85(8):2444-2448, 1988; Higgins and Sharp, Gene73(1):237-244, 1988; Higgins and Sharp, Comput. Appl. Biosci.5(2):151-3. (1989); Corpet, Nucleic Acids Res. 16(22):10881-90, 1988;Huang et al., Comput. Appl. Biosci. 8(2):155-65, 1992; and Pearson etal., Methods Mol. Biol. 25:365-389, 1994. Altschul et al. (Nature Genet.6(2):119-129, 1994) present a detailed consideration of sequencealignment methods and homology calculations.

Depending upon the extent and placement of regions of homology,homologous sequences, identified using computer-based search methodssuch as those described above, can be reasonably suspected of encodingan AlaAT. Whether such a sequence actually encodes an AlaAT can bedetermined by a number of means. As a first indicator, the annotation toa GenBank entry is used. Many sequences have been previously identifiedand tested by investigators as corresponding to AlaAT activity and theannotation to such a GenBank entry would so indicate.

Alternatively, a sequence identified from a search can be testedexperimentally to determine if it encodes an AlaAT activity. In the caseof a nucleic acid sequence that has been identified, it can be isolatedfor testing using a variety of methods known in the art. For example,the sequence of interest can be amplified by polymerase chain reaction(PCR) using primers that correspond to the 5′ and 3′ ends of thecomplementary DNA (cDNA). Such PCR methods are well known in the art andare disclosed in sources such as the laboratory manual PCR Protocols: AGuide to Methods and Applications by M. Innes, et al., Academic Press,1989. Alternatively, the desired sequence can be obtained byconventional hybridization screening using oligonucleotidescorresponding to the known nucleic acid sequence to screen a cDNAlibrary. Screening methods based on hybridization are well known in theart and are disclosed in Sambrook, Fritsch and Maniatis, MOLECULARCLONING: A LABORATORY MANUAL, 2nd edition, 1989; CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel et al., eds., 1987).

Once a DNA sequence encoding the candidate AlaAT has been obtained, itcan be cloned into a variety of expression vectors using conventionalmolecular biological methods to verify that an AlaAT has been isolated.

The AlaAT coding region can be modified in any suitable way. Forexample, it can be modified to be transcribable and translatable in theplant system; for example, the nucleotide sequence encoding the AlaATprotein can be modified such that it contains all of the necessarypoly-adenylation sequences, start sites and termination sites whichallow the coding sequence to be transcribed to mRNA and the mRNA to betranslated in the monocot plant. Further, the coding region may bemodified such that its codon usage is more similar to that of nativegenes of the monocot plant (i.e., plant optimized sequence may be used).Such nucleotide sequence modifications and the methods by which they maybe made are well known to one of skill in the art.

Many vectors for protein expression in E. coli, yeast, mammalian cells,or plants are commercially available. Expression of such a constructcontaining an AlaAT in an appropriate host cell, such as an E. coli,using a plamid such as pET vectors available from Novagen(www.Novagen.com), will reveal if the plasmid encodes an AlaAT activity.Methods for assaying for AlaAT activity are well known in the art. Onesuch method is disclosed in U.S. Pat. No. 6,084,153, which is herebyincorporated by reference in its entirety. In this method, leaf tissueis weighed and then ground with sand in a mortar and pestle inextraction buffer containing 0.1 M Tris-HCl (pH 8.5), 10 mMdithiothreitol, 15% glycerol, and 10% (w/v) PVPP. The extract isclarified by centrifugation at 6000 rpm, and the supernatant was assayedfor enzyme activity. Alanine is added to start the reaction asdescribed. See Good and Crosby, Plant Physiol. 90:1305-1309, 1989. Thisassay can be utilized for other organisms such as bacteria and yeast bysimply substituting bacteria or yeast extract for the leaf tissueextract.

Hybridization and PCR methods: Other methods can be used to isolateAlaATs that may be used in the invention. In particular, high, medium,or low stringency hybridization methods can be used to isolateorthologues or homologues of known AlaATs that maybe used in thepractice of this invention. Hybridization conditions are sequencedependent and vary according to the experimental parameters used.Generally, stringent hybridization conditions are selected to be about5° C. to 20° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Conditions fornucleic acid hybridization and calculation of stringencies can be foundin Sambrook et al. (1989) and Tijssen (Hybridization with Nucleic AcidProbes, Part II, pp. 415. Elsevier, Amsterdam, Netherlands, 1993).Examples of factors that affect nucleic acid hybridization include:temperature, salt conditions, the presence of organic solvents in thehybridization mixtures, and the lengths and base compositions of thesequences to be hybridized and the extent of base mismatching. Anexample of high stringency conditions for hybridizing a probe to afilter-bound DNA is 5×SSC, 2% sodium dodecyl sulfate (SDS), 100 ug/mlsingle stranded DNA at 55-65° C. overnight, and washing twice in 0.1×SSCand 0.1% SDS at 60-65° C. for 20 minutes.

Reduced stringency conditions can be used to isolate nucleic acidsequences that are related but have mismatches. Examples of suchconditions include lowering the hybridization and wash temperatures orraising the salt concentrations of the wash solutions. Protocols forsuch medium and low stringency hybridization methods can be found incommonly used molecular biology manuals such as the aforementionedSambrook, et al. and Ausubel, et al. references.

Other methods that can be used to isolate orthologues or homologuessuitable for use in the invention include PCR cloning. Unique ordegenerate primers can be designed to encode conserved regions in AlaATnucleotide or amino acid sequences. Such conserved regions can beidentified by aligning the sequences of known AlaATs using thealignments disclosed above. The PCR primers so designed can be used inPCR reactions to generate a portion of an AlaAT sequence from a speciesof interest which then can be used to isolate a full length cDNA byconventional library screening methods or by means of additional PCRmethods such as Rapid Amplification of cDNA Ends (RACE). Protocols forsuch PCR methods are well known in the art and can be found in sourcessuch as PCR Protocols: A Guide to Methods and Applications by M. Innes,et al., Academic Press, 1989.

An alternative strategy for identifying AlaATs for use in the inventionentails the biochemical purification of AlaATs from a source of interestbased on enzymatic activity. Because enzymatic assays for AlaAT activityare well known in the art, a skilled artisan would be able tofractionate a cell or tissue of interest and use conventionalbiochemical methods such as chromatography to purify an AlaAT tohomogeneity. Such biochemical methods are available in sources such asProtein Purification: Principles and Practice by Robert K. Scopes,Springer Advanced Texts in Chemistry, 3rd edition, 1994; Guide toProtein Purification (Methods in Enzymology Series, Vol. 182, 1990) byAbelson et al., Protein Purification Techniques: A Practical Approach(Practical Approach Series, 2001) by Simon Roe (Ed.). The AlaAT, oncepurified to homogeneity, can be used to derive partial amino acidsequences, from which oligonucleotides can be designed to clone thecorresponding cDNA by conventional molecular biological methods such aslibrary screening or PCR as described above.

FIGS. 2 and 3 and Tables 1 and 2 show alignments between AlaATs from avariety of species, ranging from E. coli to humans and including anumber of plant species. The percent homologies range from over 90% tounder 25% when the sequence of each AlaAT is compared with that of everyother AlaAT as shown in Table 1. A number of highly conserved amino acidsequences that are present in all AlaAT sequences are highlighted inblack in FIGS. 2 and 3. Such evolutionarily conserved amino acidsequences represent consensus sequences or sequence motifs that arecharacteristic of AlaATs. Frequently, such sequences form active sitesor other functionally significant regions of a protein.

TABLE 1 Barley P. miliaceum Rice Rice Rice Rice Maize ArabidopsisArabidopsis Arabidopsis AlaAT AlaAT AlaAT1 AlaAT2 AlaAT4 AlaAT3 AlaATAt1g-17290 At1g-72330 At1g-23310 Barley AlaAT 100 90 89 80 58 60 90 7778 53 P. miliaceum AlaAT 100 91 82 60 61 94 78 77 53 Rice AlaAT1 100 8259 62 91 77 76 54 Rice AlaAT2 100 57 64 81 80 80 53 Rice AlaAT4 100 4958 56 57 42 Rice AlaAT3 100 61 62 61 46 Maize AlaAT 100 77 76 52Arabidopsis 100 89 52 At1g-17290 Arabidopsis 100 51 At1g-72330Arabidopsis 100 At1g-23310 Arabidopsis At1g-70580 Capsicum AlaATChlamydomonas AlaAT Human AlaAT Yeast AlaAT E. coli AlaAT ThermococcusAlaAT Arabidopsis Capsicum Chlamydomonas Human Yeast E. coliThermococcus At1g-70580 AlaAT AlaAT AlaAT AlaAT AlaAT AlaAT Barley AlaAT52 76 51 47 46 24 24 P. miliaceum AlaAT 52 77 51 47 47 24 24 Rice AlaAT153 76 51 47 46 24 23 Rice AlaAT2 52 80 50 48 48 25 24 Rice AlaAT4 42 5742 38 38 19 19 Rice AlaAT3 46 63 46 44 42 24 22 Maize AlaAT 51 76 50 4647 23 24 Arabidopsis 51 81 50 48 44 23 23 At1g-17290 Arabidopsis 50 8249 48 45 23 24 At1g-72330 Arabidopsis 93 51 67 46 44 24 26 At1g-23310Arabidopsis 100 51 66 45 45 24 26 At1g-70580 Capsicum AlaAT 100 50 48 4623 24 Chlamydomonas AlaAT 100 47 42 25 26 Human AlaAT 100 44 22 25 YeastAlaAT 100 19 24 E. coli AlaAT 100 45 Thermococcus AlaAT 100

TABLE 2 Arabi- Arabi Arabi Arabi dopsis dopsis dopsis dopsis Barley RiceRice Rice Rice At1g- At1g- At1g- At1g- AlaAT P. miliaceum AlaAT1 AlaAT2AlaAT4 AlaAT3 Maize 17290 72330 23310 70580 Capsicum Barley AlaAT 100 9088 80 57 58 90 76 77 51 50 75 P. miliaceum 100 91 82 59 60 94 77 77 5251 77 Rice AlaAT1 100 82 58 60 90 76 76 53 52 76 Rice AlaAT2 100 56 6380 80 80 51 50 80 Rice AlaAT4 100 48 57 54 56 41 40 56 Rice AlaAT3 10060 61 60 44 44 62 Maize 100 76 75 51 50 76 Arabidopsis 100 89 50 50 81At1g17290 , Arabidopsis 100 49 49 82 At1g72330 Arabidopsis 100 93 50At1g23310 Arabidopsis 100 50 At1g70580 Capsicum 100Overexpression of AlaATs in Monocot Plants

Once an AlaAT has been identified and verified as corresponding to abona fide AlaAT, a construct for overexpression of the AlaAT in amonocot plant of interest is generated using methods well known in theart. A variety of plasmids are available for this purpose as disclosedbelow. A variety of promoters such as constitutive promoters, variousinducible promoters, or tissue-specific promoters can be used forexpression.

Promoters

The promoters suitable for use in the constructs of this invention arefunctional in monocot plants and in host organisms used for expressingthe constructs described. Many plant promoters are publicly known andseveral examples are listed below. These include constitutive promoters,inducible promoters, tissue- and cell-specific promoters anddevelopmentally regulated promoters. Methods are disclosed below for theselection of promoters that are suitable for use in practicing theinvention.

Promoters can be isolated by procedures well known in the art of plantmolecular biology. Exemplary, but non-limiting, promoters that can beused in the practice of this invention include: the rice antiquitin(OsAnt1) promoter, which is described in Example 1 below, as well asother antiquitin promoters, as described in Example 5 below; the riceactin 1 (Act-1) promoter, which is described in U.S. Pat. No. 5,641,876;the maize ubiquitin-1 (Ubi-1) promoter, which is described in U.S. Pat.Nos. 5,510,474, 6,054,574, and 6,977,325; the maize alcoholdehydrogenase-1 (Adh1) promoter, which is described in Kyozuka et al.,Mol. Gen. Genet. 228(1-2): 40-48, 1991; and the CaMV 35S and 19Spromoters, which are described in U.S. Pat. No. 5,352,605. For otherpromoters useful in monocots, see: cambia.org website.

One type of promoter particularly useful for expression of a target genesuch as AlaAT in a plant is a monocot antiquitin promoter. The riceantiquitin promoter is described in Example 1. Other antiquitinpromoters are described in Example 5. Knowing the monocot antiquitinpromoters disclosed in these Examples, one of skill could readilyidentify other monocot antiquitin promoters using methods similar tothose described in Example 1 for identification of the rice antiquitinpromoter using the btg 26 gene. For example, the sequence can be subjectto analysis with a promoter prediction software such as the TSSP plantpromoter prediction software found at the softberry.com website toidentify likely TATA box sequences and other promoter sequence elementsand further analyzed for promoter motifs that may be recognition sitesfor transcription factors using Signal Scan Software (Prestridge, 1991;available at bimas.dcrt.nih.gov/molbio/signal).

Sequences likely to encode promoters can be confirmed by synthesizingvarious fragments and testing for expression or introducing pointmutations in certain regions and testing for loss of activity using anyassay system known to those of skill in the art as being useful formeasuring the promoter activity, such as expression of a reporter geneunder the control of a promoter sequence. Reporter genes can be anypolynucleotide the transcription of which under the control of apromoter sequence, the subsequent translation thereof, or both, can bereadily detected by a skilled artisan. The reporter gene does not haveto encode a full length protein. In some instances, the reporter genecan even be an oligonucleotide. Most commonly, the reporter gene encodesa protein with detectable activity. Common reporter genes include GUS,luciferase, GFP, beta-galactosidase, CAT, alkaline phosphatase, etc. Inpreferred embodiments, the reporter gene is GUS.

The expression of the reporter gene can be measured at either the mRNAor protein level using any method known to those of skill in the art.For example, mRNA levels can be detected using a cell-free transcriptionassay. Alternatively, protein levels can be measured by detecting enzymeactivity, using antibodies specific for the protein, or atranscription-translation assay, which allows detection of both the mRNAlevel and the protein or peptide level.

Promoters from genes that are regulated similarly to the antiquitingenes in plants might also find use in the invention. These genes couldbe turgor responsive genes that are expressed in root tissues and couldbe induced by ABA and/or under stress conditions such as drought andsalt.

Transformation Methods

After a suitable construct has been made, transgenic plants of interestcan be generated using transformation methods well known in the art anddescribed herein as well as in the Examples below. An exogenous nucleicacid molecule can be introduced into a monocot plant for ectopicexpression using a variety of transformation methodologies includingAgrobacterium-mediated transformation and direct gene transfer methodssuch as electroporation and microprojectile-mediated transformation(see, generally, Wang et al. (eds), Transformation of Plants and SoilMicroorganisms, Cambridge, UK: University Press, 1995, which isincorporated herein by reference). Transformation methods based upon thesoil bacterium, Agrobacterium tumefaciens, are particularly useful forintroducing an exogenous nucleic acid molecule into a seed plant. Thewild-type form of Agrobacterium contains a Ti (tumor-inducing) plasmidthat directs production of tumorigenic crown gall growth on host plants.Transfer of the tumor-inducing T-DNA region of the Ti plasmid to a plantgenome requires the Ti plasmid-encoded virulence genes as well as T-DNAborders, which are a set of direct DNA repeats that delineate the regionto be transferred. An Agrobacterium-based vector is a modified form of aTi plasmid, in which the tumor inducing functions are replaced by thenucleic acid sequence of interest to be introduced into the plain host.

Agrobacterium-mediated transformation generally employs cointegratevectors or, preferably, binary vector systems, in which the componentsof the Ti plasmid are divided between a helper vector, which residespermanently in the Agrobacterium host and carries the virulence genes,and a shuttle vector, which contains the gene of interest bounded byT-DNA sequences. A variety of binary vectors are well known in the artand are commercially available, for example, from Clontech (Palo Alto,Calif.). Methods of co-culturing Agrobacterium with cultured plant cellsor wounded tissue such as root explants, hypocotyledons, stem pieces ortubers, for example, also are well known in the art (Glick and Thompson,Methods in Plant Molecular Biology and Biotechnology. CRC Press, BocaRaton, Fla., pp 179-20519, 1993). Wounded cells within the plant tissuethat have been infected by Agrobacterium can develop organs de novo whencultured under the appropriate conditions; the resulting transgenicshoots eventually give rise to transgenic plants that ectopicallyexpress a nucleic acid molecule encoding an AlaAT protein. Agrobacteriumalso can be used for transformation of whole seed as described inBechtold et al., C. R. Acad. Sci. Paris. Life Sci. 316:1194-1199, 1993,(which is incorporated herein by reference). Agrobacterium-mediatedtransformation is useful for producing a variety of transgenic seedplants (Wang et al., supra, 1995).

Microprojectile-mediated transformation also can be used to produce atransgenic plant that ectopically expresses AlaAT. This method, firstdescribed by Klein et al. (Nature 327:70-73, 1987, which is incorporatedherein by reference), relies on microprojectiles such as gold ortungsten that are coated with the desired nucleic acid molecule byprecipitation with calcium chloride, spermidine or PEG. Themicroprojectile particles are accelerated at high speed into a planttissue using a device such as the BIOLISTIC PD-1000 (Biorad, Hercules,Calif.).

Microprojectile-mediated delivery or “particle bombardment” isespecially useful to transform plants that are difficult to transform orregenerate using other methods. Microprojectile-mediated transformationhas been used, for example, to generate a variety of transgenic plantspecies, including cotton, tobacco, maize, hybrid poplar and papaya (seeGlick and Thompson, supra, 1993) as well as cereal crops such as wheat,oat, barley, sorghum and rice (Duan et al., Nature Biotech. 14:494-498,1996; Shimamoto, Curr. Opin. Biotech. 5:158-162, 1994; each of which isincorporated herein by reference). In view of the above, the skilledartisan will recognize that Agrobacterium-mediated ormicroprojectile-mediated transformation, as disclosed herein, or othermethods known in the art can be used to produce a transgenic seed plantof the invention.

Alternative gene transfer and transformation methods useful in theinvention include, but are not limited to, liposomes, electroporation orchemical-mediated uptake of free DNA, calcium phosphate co-precipitationtechniques, and micro- or macroinjection, direct DNA transformation, andmay involve Ti plasmids, Ri plasmids, or plant virus vectors. Suchtransformation methods are well documented in the art.

Growth and NUE Assays

The resulting transgenic plant of interest are tested for expression ofthe AlaAT transgene and those plant lines that express the AlaATtransgene are tested for the effect of the expressed transgene on plantgrowth or nitrogen utilization. Suitable tests for monocot plant growthcan include a variety of assays such as measuring plant height, seedweight, stem diameter, number of plant leaves, plant biomass as measuredin fresh weight or dry weight of roots, leaves, shoots, buds, andflowers, to name but a few such measurement parameters. Tests for NUEcan include growth of transgenic plants under different suboptimalnitrogen conditions. Tests may be field test, greenhouse or growthchamber tests or in vitro tests. Plants may be grown hydroponically inPerlite™, other commercially available growing material, soil, or inagar-based media.

Use of Monocot Antiquitin Promoters to Direct Expression of Other CodingRegions

Monocot antiquitin promoters can also be used to direct expression ofcoding regions other than AlaAT.

The coding region of interest, or target gene, operatively linked to themonocot antiquitin promoter may be any nucleotide sequence that isdesirably expressed within a plant. General classes of coding regionswhich may be advantageously employed in the methods and constructs ofthe invention include nucleotide sequences encoding structural proteins;proteins involved in the transport of nitrogen; proteins involved in theuptake of nitrogen; proteins involved in both the transport and uptakeof nitrogen; enzymes and proteins involved in nitrogen utilization;proteins involved in plant resistance to pesticides or herbicides;proteins involved in plant resistance to nematodes, viruses, insects, orbacteria; proteins involved in plant resistance to stress, for examplebut not limited to osmotic, temperature, pH, or oxygen stress; proteinsinvolved in stimulation or continuation of plant growth; proteinsinvolved in phytoremediation; or proteins having pharmaceuticalproperties or encoding enzymes which produce compounds havingpharmaceutical properties.

For example, the coding region of interest may encode a nitrogenutilization protein and, in particular, an enzyme that assimilatesammonia into amino acids or uses the formed amino acids in biosyntheticreactions. This protein may be selected from, but not limited to, anitrate transporter (high or low affinity), an ammonium transporter, anammonia transporter, an amino acid transporter, alanine dehydrogenase,glutamine synthetase (GS), asparagine synthetase (AS), glutamatesynthase (also known as glutamate 2:oxogluturate amino transferase andGOGAT), asparaginase (ANS), glutamate dehydrogenase (GDH), nitratereductase, aspartate aminotransferase (AspAT), AlaAT, and other knownaminotransferases. Such proteins are disclosed in US Patent ApplicationPublication Number 2005/0044585, which is hereby incorporated byreference in its entirety.

The target gene or coding region of interest may be naturally expressedin the plant or it may be heterologous to the plant. The gene mayoriginate from any source, including viral, bacterial, plant or animalsources. Preferably, the coding region of interest is heterologous tothe monocot antiquitin promoter sequence to which it is operativelylinked, in that it is not from the gene the monocot antiquitin promotersequence is naturally linked to.

The coding region can be modified in any suitable way in order toengineer a gene with desirable properties. The coding region can bemodified to be transcribable and translatable in the plant system; forexample, the nucleotide sequence encoding the protein of interest can bemodified such that it contains all of the necessary poly-adenylationsequences, start sites and termination sites which allow the codingsequence to be transcribed to mRNA (messenger ribonucleic acid) and themRNA to be translated in the plant. Further, the coding region may bemodified such that its codon usage is more similar to that of nativegenes of the plant (i.e., plant optimized sequence may be used). Suchnucleotide sequence modifications and the methods by which they may bemade are well known to one of skill in the art.

The methods and constructs described herein allow the production ofplants and seeds having expression of one or more desired genes in theplant. There is a wide variety of possible applications of the plantsdescribed herein, including, but not limited to, the production ofplants having increased stress tolerance, improved nitrogen uptake,improved nitrogen utilization, improved nutrient content, improvednutrient yields of desired compounds, and phytoremediative properties.Specific applications are further described below.

The following examples further demonstrate several preferred embodimentsof this invention. While the examples illustrate the invention, they arenot intended to limit the invention.

EXAMPLES Example 1 Demonstration of NUE in Rice Expressing Barley AlaAT

Identification and Characterization of a Rice Antiquitin Promoter(OsAnt1)

The nucleotide sequence (bp 366-3175) of the btg26 gene (Stroeher etal., Plant Mol. Biol. 27:541-551, 1995; accession number S77096) wasused to search the nucleotide database at NCBI using the blastn searchtool. A rice sequence (accession number AF323586) was identified andthis nucleotide sequence was used to search the TIGR Oryza sativasequencing project (tigr.org/tdb/e2k1/osa1/). The rice homologue ofbtg26, Oryza sativa antiquitin (OsAnt1), was identified on chromosome 9of rice (accession number AP005570; 100216-91996 base pairs). A 973-bpsequence (nucleotides 101189-100216 of AP005570) upstream of the startcodon of OsAnt1 is shown in FIG. 4 (SEQ ID NO:1).

The sequence of the 403 bps upstream (5′) of the ATG start codon of theOsAnt1 gene was selected for further analysis. To determine if thesequence was likely to function as a promoter sequence, the sequence wasanalyzed using the TSSP plant promoter prediction software found at thesoftberry.com website. The analysis predicted that the sequence was aplant promoter sequence. The most likely location of the TATA box (boldin FIG. 4), as well as other promoter sequence elements, was determined.

Since the projected OsAnt1 promoter sequence was predicted to containpromoter elements according to the Softberry analysis, the sequenceswere analyzed for promoter motifs that may be recognition sites fortranscription factors using Signal Scan Software (Prestridge, ComputAppl Biosci 7(2):203-6, 1991; at bimas.dcrt.nih.gov/molbio/signal). Fivedifferent signal sequences were predicted in the OsAnt1 promoter,including ADR1, DBF-A, GAL4, HSTF and RAF transcription factor bindingsites.

The OsAnt1 sequence was compared to nucleic acid sequences of btg26promoter sequences from Brassica napus and Arabidopsis using theClustalW 1.8 multiple sequence alignment software on the BCM SearchLauncher homepage (searchlauncher.bcm.tmc.edu/) and BOXSHADE server(ch.embnet.org/software/BOX_form.html). Inspection of conservednucleotides revealed that the Brassica and Arabidopsis turgor gene-26promoter sequences are more similar to each other than to the OsAnt1sequence. A feature among all three promoter sequences (rice, Brassica,Arabidopsis) is the polypyrimidine (CT) tracts evident within thenucleotide sequences. These tracts range from 20-22 bases and are foundjust upstream of the probable TATA boxes in all three promotersequences. Furthermore, the OsAnt1 sequence has a second polypyrimidinetract just upstream of the ATG start codon.

Cloning of a Rice Antiquitin Promoter

Rice genomic DNA was isolated from cv. Kitaake. The following PCRprimers (positions underlined in FIG. 4) corresponding to the OsAnt1promoter region were selected:

(SEQ ID NO: 2) Primer 1: AGGAAGTGATTTTTAGCGTAGCTG; (SEQ ID NO: 3)Primer 2: ATGGCAGAAGAGAGAGAGAGAGAGG.

Touch-down PCR was conducted using rice genomic DNA and the aboveprimers. A 975-bp fragment was produced. The amplified PCR fragment wasligated into pCR®II-TOPO vector (Invitrogen) and transformed into E.coli, TOP 10 cells. The resulting plasmid is designatedpT-riceOsAnt1pro.

Sequence analysis indicated that the 975-bp PCR fragment encodes apromoter sequence designated the OsAnt1 promoter sequence. Comparison ofthe OsAnt1 promoter from cv. Kitaake with that of cv. Nipponbare(obtained from the database) revealed that they share 99.9% identity.The putative TATA box was found 145-bps upstream of the start codon.

Production of the OsAnt1pro-GUS Construct

The beta-glucuronidase (GUS) reporter gene driven by OsAnt1 was producedusing the steps shown schematically in FIG. 5. The RiceOsAnt1pro-GUSconstruct was produced by amplifying the pT-RiceOsAnt1pro template usingthe following primers:

Primer 3: EcoRI-OsAnt1 promoter sequence (SEQ ID NO: 4)GGAATTCAGGAAGTGATTTTT Primer 4: NcoI-OsAnt1 promoter sequence(SEQ ID NO: 5) CATGCCATGGATGGCAGAAGA

The resultant PCR fragments were ligated into the plant binary vector,pCAMBIA1305.1, digested with EcoR1 and Nco1 to produce apCAMBIA1305.1-riceOsAnt1pro-GUS construct. The EcoRI and NcoI sequencesat the end of primers 3 and 4, respectively, allowed insertion of thePCR fragment into the pCAMBIA1305.1 vector, replacing the existingCaMV35s promoter with the OsAnt1 promoter sequence. The NcoI sequence(CCATGG) includes a Met codon, ATG, which is in frame with the GUSreporter gene and allows expression of the GUS reporter gene from theOsAnt1 promoter sequence.

Production of the OsAnt1pro-AlaAT Construct

The barley AlaAT gene driven by OsAnt1 was produced using the stepsshown schematically in FIG. 6. The RiceOsAnt1pro-AlaAT construct wasproduced by amplifying the pT-RiceOsAnt1 pro template using thefollowing primers:

Primer 3: EcoRI-OsAnt1 promoter sequence (SEQ ID NO: 4)GGAATTCAGGAAGTGATTTTT Primer 5: PstI-OsAnt1 promoter sequence(SEQ ID NO: 6) AACTGCAGATGGCAGAAGA

The resultant PCR fragments, digested with EcoR1 and Pst1, were ligatedinto the plant binary vector, pCAMBIA1300, and digested with EcoR1 andPst1 to produce pCAMBIA1300-riceOsAnt1pro.

An AlaAT DNA fragment was amplified by PCR using pAG001 as a template.pAG001 is described in U.S. Pat. No. 6,084,153 where it is identified aspbtg26/AlaAT/nos. It contains the btg26 promoter linked to the barleyAlaAT gene with a nopaline synthase terminator. The barley AlaAT/nosterminator sequences were amplified from pAG001 using the followingprimers:

Primer 6: PstIAlaAT sequence AACTGCAGATGGCTGCCACCG (SEQ ID NO: 7)Primer 7: HindIII-NOS terminator sequence CCCAAGCTTCCCGATCTAGTA(SEQ ID NO: 8)

The resulting AlaAT/nos fragment was digested with Pst and HindIII andligated into the pCAMBIA1300-riceOsAnt1pro digested with Pst1 andHindIII to produce a pCAMBIA1300-riceOsAnt1pro-AlaAT construct.

Transformation of Rice

Rice transformation methods are well known in the art (Sridevi et al.,Current Sci. 88:128-132, 2005; Saharan et al., African J. Biotech3(11):572-575, 2004; Khanna et al., Aust. J. Plant Physiol. 26:311-324,1999; Zhang et al., Molecular Biotechnology 8(3):223-231, 1988; Rashidet al., Plant Cell Rep. 15:727-730, 1996; Aldemita and Hodges, Planta199:612-617, 1996; Hiei et al., Plant J. 6:271-282, 1997; Li et al.,Plant Cell Rpt 12:250-255, 1993; Christou et al., Biotechnology9:957-962, 1991). Agrobacterium-mediated transformation of rice wascarried out as modified from U.S. Pat. No. 5,591,616 as described below.

pCAMBIA1305.1-riceOsAnt1pro-GUS and pCAMBIA1300-riceOsAnt1pro-AlaAT weretransferred into Agrobacterium strain EHA105 (Hood et al., TransgenicRes. 2: 208-218, 1993) by electroporation (Sambrook et al., supra,1989). Agrobacterium cells were plated on solid AB medium (Chilton etal., Proc. Natl. Acad. Sci. USA 71:3672-3676, 1974) containing 50 mg/lkanamycin and incubated at 28° C. for 3 days. The bacteria were thencollected with a flat spatula and resuspended in liquid co-cultivationmedium (R2-CL, Table 3) by gentle vortexing prior to transforming therice tissues.

Mature seeds of rice (Oryza sativa L. cv. Nipponbare) were used in thetransformation experiment. The seeds were dehusked and surfacesterilized by dipping (1 min) in 70% (v/v) ethanol followed by soakingin 50% bleach plus 0.1% Tween-20 for 10 min and then rinsing five timesin sterile distilled water. Following sterilization, seeds were culturedon callus induction medium (NB, Table 3) and incubated for three weeksin the dark at 28° C.

TABLE 3 Medium used for callus induction, inoculation, co-culture,resting phase, selection, regeneration and rooting Medium CompositionNB^(a) N6 major salt and iron source (Chu (1975) Sci. Sin. 5: 659-Callus induction medium 668) + B5 major salts and vitamins (Gamborg etal. (1968) (filter sterilize) Exp. Cell Res. 50: 151-158) + 3AA (100mg/l L-tryptophan + 500 mg/l L-proline + 500 mg/l L-glutamine) + 500mg/l casein hydrolysate + 2.0 mg/l 2,4-D + 0.5 mg/l picloram + 30 g/lsucrose, pH 5.8, 0.3% gelrite R2-CL R2 major and minor salts, vitaminsand iron source without Liquid co-culture medium sucrose (Ohira et al.(1973) Plant and Cell Physiol. 14: (filter sterilize) 1113-1121) + 0.25Mglucose + 125 μM acetosyringone + 10 mM MES buffer, pH 5.2 + 50 mMpotassium phosphate buffer, pH 5.2 + 400 mg/l L-cysteine + 2.0 mg/l2,4-D + 0.5 mg/l picloram + 0.5 mg/l BAP, pH 5.2 R2-CS R2 major andminor salts, vitamins and iron source without Solid co-culture mediumsucrose (Ohira et al. (1973) Plant and Cell Physiol. 14: (filtersterilize) 1113-1121) + 0.25M glucose + 125 μM acetosyringone + 10 mMMES buffer, pH 5.2 + 50 mM potassium phosphate buffer, pH 5.2 + 400 mg/lL-cysteine + .2.0 mg/l 2,4-D + 0.5 mg/l picloram + 0.5 mg/l BAP, pH5.2 + 0.3% gelrite R2-AS R2 major and minor salts, vitamins and ironsource without Resting phase sucrose + 0.25M sucrose + 0.5 mMacetosyringone + 10 mM (filter sterilize) MES buffer, pH 5.0 + 50 mMpotassium phosphate buffer, pH 5.0 + 10 mM CaCl₂ + 400 mg/l L-cysteine +2.0 mg/l 2,4-D + 0.5 mg/l picloram + 0.5 mg/l BAP + 250 mg/lcefotaxime + 250 mg/l amoxicillin, pH 5.0, 0.3% gelrite R2S R2 major andminor salts, vitamins and iron source + 30 g/l Selection mediumsucrose + 2.0 mg/l 2,4-D + 0.5 mg/l picloram + 50 mg/l (filtersterilize) hygromycin + 250 mg/l cefotaxime + 100 mg/l amoxicillin, pH5.8, 0.3% gelrite NBS NB medium + 3AA + 2.0 mg/l 2,4-D + 0.5 mg/lPicloram + Selection medium-II 50 mg/l hygromycin + 250 mg/lcefotaxime + 100 mg/l (filter sterilize) amoxicillin, pH 5.8, 0.3%gelrite PRN NB medium + 3AA + 5 mg/l ABA + 2 mg/l BAP + 0.5 mg/lPre-regeneration medium NAA + 50 mg/l hygromycin + 100 mg/l cefotaxime +(filter sterilize) 50 mg/l amoxicillin, pH 5.8, 0.4% gelrite RN NBmedium + 3 mg/l BAP + 0.5 mg/l NAA + 50 mg/l Regeneration mediumhygromycin + 100 mg/l cefotaxime + 50 mg/l amoxicillin, (filtersterilize) pH 5.8, 0.4% gelrite R Rooting medium ½MS (Murashige andSkoog (1962) Physiol. Plant 15: (Autoclave/filter sterilize) 473-497) +50 mg/l hygromycin^(b) + 100 mg/l cefotaxime + 50 mg/l amoxicillin, pH5.8, 0.3% gelrite ^(a)NB medium with 1.25 mg/l CUSO₄ ^(b)Optional

After three weeks, 3-5 mm long embryogenic nodular units released fromthe scutellum-derived callus at the explant/medium interface wereimmersed into 25 ml of liquid co-culture medium (R2-CL, Table 3)containing Agrobacterium cells at the density of 3-5×10⁹ cells/ml(OD₆₀₀=1) in a 100 mm-diameter Petri dish for 10-15 minutes. Embryogenicunits were then blotted dry on sterilized filter paper, transferred to aPetri dish containing solid co-culture medium (R2-CS, Table 3) andincubated for three days at 25° C. in the dark. Co-cultured embryogeniccalli were then transferred to resting medium (R2-AS, Table 3) andincubated at 28° C. in the dark for a week.

After a week, uncontaminated embryogenic units were then individuallytransferred to selection medium (R2S, Table 3) containing hygromycin forselection of transformed tissue and incubated at 28° C. in the dark.Following 3 weeks of selection on R2S medium, the embryogenic units thatturned dark brown with brownish protuberances arising throughout thecallus surface were transferred to NBS selection medium (Table 3). After5 weeks of co-culture, the protuberances developed into brownishglobular structures that were gently teased apart from callus andincubated for 2 weeks in the resealed Petri dish. After 2 weeks, theseglobular structures converted into round shaped, compact and yellowishcalli.

The putatively transgenic, hygromycin-resistant calli were gently pickedout, transferred, cultured on pre-regeneration medium (PRN, Table 3) andthen incubated for a further week. All of the resistant callioriginating from a single co-cultured embryogenic nodular unit weregrouped in a sector of the PRN dish. Creamy-white, lobed calli with asmooth and dry appearance were individually transferred to regenerationmedium (RN, Table 3), incubated for 2 days in the dark, then maintainedfor three weeks under a 12/12-h (day/night) photoperiod with lightprovided at an intensity of 55 μmol/m per sec. Green shoots regeneratingfrom a resistant callus were dissected and sub-cultured in test tubecontaining rooting medium (R, Table 3) for 1-2 weeks to promote vigorousroots and tillers before being transferred to pots in growth rooms.Transgenic plants were grown to maturity in 16-cm pots containingsoil-less potting mixture (Metromix 220). Plants were maintained ingrowth rooms set to 28° C. and 14/10 hours day/night photoperiods.Fertilizer was applied twice a week starting two weeks after planting inpots. The fertilizer mix contained 225 g 20/20/20 fertilizer, 50 g ofplant micronutrients, 6.1 g of CuSO₄.5H₂O, 140 g FeEDTA, 13.8 gZnSO₄.7H₂O, 260 g MgSO₄.7H₂O, 3.7 g H₃BO₃ for a total of 712.4 g. Twograms of the fertilizer mix are dissolved in 8 liters of water andapplied twice a week to 24 plants.

Analysis of Expression Directed by the OsAnt1 Promoter Sequence

Induction of expression directed by the OsAnt1 promoter sequence wasexamined using rice plants transformed with the OsAnt1pro-GUS construct.Plants were germinated and grown hydroponically in sterile conditions inMagenta jars. Two-week-old plants were stained for in vivo GUS activityby injecting into the root media 5 mls of 50 mM phosphate buffer (pH7.5) containing 0.2 mM X-gluc(5-bromo-4-chloro-3-indolyl-beta-glucuronic acid) and incubating theplants in this media for 1-24 hours. Root tissue was then viewed under adissection microscope and photographs were taken, which are shown inFIG. 7.

Dark stained areas in FIG. 7 indicate expression of the GUS reportergene. There is no expression of the GUS reporter gene driven by theOsAnt1 promoter in the root tip (specifically the dividing cells);however, expression begins very quickly in the cell expansion zone, justbehind the root tip. The OsAnt1 promoter sequence directed expression ofthe GUS reporter gene in the root hairs as well. Further from the roottip in more mature roots, expression is lost from the main root, butlateral roots stain very heavily, indicating that OsAnt1 directsexpression in these lateral roots very strongly.

Analysis of Transformed Rice Plants Containing the AlaAt Construct

Fifty-eight OsAnt1/AlaAT/NOS transgenic plants were generated andmeasurements for flowering, tiller number, seed weights and biomass atmaturity were recorded for the T₀ generation plants.

The dry weight biomass of OsAnt1/AlaAT plants and control plants wasmeasured at maturity, and the data is presented in FIG. 8. The averagebiomass of the transgenic OsAnt1/AlaAT plants was higher than theaverage biomass of control plants.

Seeds were collected from OsAnt1/AlaAT plants and control plants atmaturity and the total weight of the seeds was measured. The results areshown in FIG. 9, which shows that the total seed weight of seedscollected from OsAnt1/AlaAT plants was higher than that of the seedweight from control plants.

FIG. 10 shows the relationship between dry weight biomass and total seedweight for each transgenic plant. A substantially linear correlation isshown, which indicates that an increase in biomass results in acorresponding increase in total seed weight in OsAnt1/AlaAT plants.

These results indicate that OsAnt1/AlaAT transgenic plants are capableof optimizing the utilization of available nutrients thereby resultingin an increase in plant biomass, seed yield or a combination thereof.

Example 2 Demonstration of NUE in Maize using OsAnt1/Barley AlaAT

The OsAnt1-pro-AlaAT construct can be incorporated into suitable plantbinary vectors for use in Agrobacterium-mediated transformation ofmaize. Many methods for transformation of immature embryos of maizeusing a variety of selectable markers are known in the art (Ishida etal., Nature Biotech. 14:745-750, 1996; Lupotto, Maydica 44:211-218,1999; Zhao et al.; Molec. Breeding 8:323-333, 2001; Frame et al., PlantPhysiol. 129:13-22, 2002 and Miller et al., Transgenic Res. 11:381-396,2002, U.S. Pat. No. 5,591,616. Contract production of transgenic maizeplants is also available through facilities such as the PlantTransformation Facility, Iowa State University, Ames, Iowa.

Alternatively, the OsAnt1pro-AlaAT sequence can be used similarly inbiolistic transformation methods for maize (Wright et al., Plant CellReports 20(5):429-436, 2001; Brettschneider et al., Theoret. Appl.Genet. 94:737-748, 1997; Gordon-Kamm et al., Plant Cell 2(7):603-618,1990; Fromm et al., Biotechnology (NY). 8(9):833-9. 1990).

Maize plants can be tested for NUE by measurement of biomass and seedyield during growth under various nitrogen fertilizer regimes includinglimiting nitrogen. Plant biomass can be fresh weight or dry weight,total plant weight, leaf weight or root weight. Suboptimal nitrogenconditions are those conditions in which nitrogen concentrations limitgrowth. Under such conditions, addition of added nitrogen such asfertilizer will increase growth. For each of these tests, biomass andseed yield can be evaluated in growth chamber, greenhouse or fieldtests.

Example 3 Demonstration of NUE in Wheat using OsAnt1/Barley AlaAT

Similar to maize, the OsAnt1-pro-AlaAT construct can be used forparticle-gun bombardment transformation methods of wheat (Pastori etal., J. Exp. Bot. 52(357):857-863, 2001; Becker et al., Plant J.5:299-307, 1994) or incorporated into suitable plant binary vectors foruse in Agrobacterium-mediated transformation of wheat (Cheng et al.,Plant Physiol. 115:971-980, 1997; U.S. Patent ApplicationUS2003/0024014A1) Other methods for wheat transformation are establishedin the art.

Wheat plants can be tested for NUE by measurement of biomass and seedyield during growth under various nitrogen fertilizer regimes includinglimiting nitrogen. Plant biomass can be fresh weight or dry weight,total plant weight, leaf weight or root weight. Suboptimal nitrogenconditions are those conditions in which nitrogen concentrations limitgrowth. Under such conditions, addition of added nitrogen such asfertilizer will increase growth. For each of these tests, biomass andseed yield can be evaluated in growth chamber, greenhouse or fieldtests.

Example 4 Demonstration of NUE in Sorghum using OsAnt1/Barley AlaAT

Agrobacterium-mediated sorghum transformation of immature embryos with abinary vector containing any of the OsAnt promoter/AlaAT constructs canbe achieved according to methods established in the art (Zhao et al.,Plant Mol. Biol. 44(6):789-98, 2000; Gao et al., Genome 48(2):321-33,2005; Zhao, Z. Y., Methods Mol, Biol. 343:233-44, 2006; Howe et al.,Plant Cell Rep. 25(8):784-91, 2006).

Sorghum plants can be tested for NUE by measurement of biomass and seedyield during growth under various nitrogen fertilizer regimes includinglimiting nitrogen. Plant biomass can be fresh weight or dry weight,total plant weight, leaf weight or root weight. Suboptimal nitrogenconditions are those conditions in which nitrogen concentrations limitgrowth. Under such conditions, addition of added nitrogen such asfertilizer will increase growth. For each of these tests, biomass andseed yield can be evaluated in growth chamber, greenhouse or fieldtests.

Example 5 Identification of Alternate (Antiquitin) Promoter Sequencesfor Use in NUE Constructs

Other antiquitin promoter sequences useful in monocots can be identifiedin sequence databases. As described for isolation of the rice promoterin Example 1, the nucleotide sequence (bp 366-3175) of the btg26 gene(Stroeher et al., Plant Mol. Biol. 27:541-551, 1995; accession numberS77096) is used to search the nucleotide database at NCBI using theblastn search tool. In addition to the rice sequence identified, othermonocot antiquitin sequences are identified in the nr database includingsorghum (accession number U87982), maize (accession numbers AY103614 andBT017791), cocoa (Theobroma cacao; accession number DQ448866; andCurculigo latifolia, accession number X64110). ESTs for wheat, sugarcaneand switchgrass can also be identified in databases using the identifiedrice antiquitin nucleotide or amino acid sequences using various searchalgorithms.

Similar to the identification of the OsAnt1 promoter, a sorghum promotersequence was identified by using the rice nucleotide sequence of theantiquitin clone (accession number AF323586) in a BLAST search of thesorghum sequences in the NCBI Genome Sequence Survey (gss) Database.Clone CW033386 was identified as containing 443 nucleotides of sequenceupstream of the ATG start codon of a sorghum antiquitin gene (SEQ IDNO:9, FIG. 11). This sequence can be used as a promoter sequence aloneor methods to clone and sequence larger genomic fragments can be used toidentify sequences further upstream. These fragments can be parts of BACsequences or from further genome sequencing efforts in sorghum or thelike. One skilled in the art could also walk-up the genome using methodssuch as inverse PCR and genome walking kits.

An upstream sequence of the maize antiquitin gene was identified in aBLAST search using the sequence of the rice antiquitin clone against theZea mays sequences in the NCBI Genome Survey Sequences Database.Accession BH215004 was identified as containing a 204-bp sequenceupstream of a maize antiquitin gene (SEQ ID NO:10, FIG. 12). Thissequence can be used as a promoter sequence alone or methods to cloneand sequence larger genomic fragments can be used to identify sequencesup to 1.5 kb upstream of this particular antiquitin gene. Sequencesincluding the longer promoters could be used to design promoter/AlaATgene constructs as described below.

Example 6 Construction of Alternate Expression Cassettes for NUEConstructs

Promoter cassettes for expression of various genes are constructed bycombining the promoter of interest with a nos terminator with convenientrestriction sites in between the promoter and terminator for genecloning. Other restriction sites flank the promoter and terminator tofacilitate movement of the cassette to a binary vector for planttransformation.

A base vector containing the nos terminator is constructed by PCRamplifying the nos region contained in the binary vectors described inU.S. Pat. No. 6,084,153 with the primers NOSupper2:5′-CCTAGGCCATGGTTCAAACATTTGGCAATAAAGTTT-3′ (SEQ ID NO: 11) and NOSlower:5′-TTAATTAACGATCTAGTAACATAGATGACA-3′ (SEQ ID NO: 12). NOSupper2 suppliesAvrII and NcoI restriction sites at the 5′-end of the nos terminator andNOSlower supplies a Pac1 site at the 3′ end of the amplified fragment.PCR was performed using the BD Advantage™ 2 PCR kit followingmanufacturer's instructions. The resulting 263 bp fragment is clonedinto pCR®2.1-TOPO® vector using a TOPO TA Cloning® Kit (Invitrogen) andOne Shot® E. coli cells following manufacturer's instructions. Thisplasmid is Nos/PCR2.1.

The Nco1 site in the kanamycin resistance gene in the Nos/pCR2.1backbone is removed using the QuikChange® XL Site-Directed MutagenesisKit (Stratagene) following manufacturer's instructions. Primers that maybe used to introduce a silent nucleotide change are NcoIpCR2.1 Lower5′-GCAGGCATCGCCATGAGTCACGACGAGATC-3′ (SEQ ID NO: 13) and NcoIpCR2.1Upper 5′-GATCTCGTCGTGACTCATGGCGATGCCTGC-3′ (SEQ ID NO: 14). Deletion ofthe Nco1 site may be verified by restriction analysis and growth of theE. coli on kanamycin. This resulting plasmid is Nos/pCR2.1mut.

An alternative expression cassette for expressing genes from the OsAnt1promoter is made in the following manner. The OsAnt1 promoter is clonedfrom rice var. Nipponbare genomic DNA (made by manufacturer'srecommendation, Sigma Extract-n-AMP™) using PCR. Primers for a slightlylonger version of the OsAnt1 promoter than that shown in SEQ ID NO: 1are:

Forward primer (SEQ ID NO: 15) 5′-ATTAAACCTAGGTTAATTAAGTTTAAACGACCTATAAAGTCAAATGCAAA T- 3′ andreverse primer (SEQ ID NO: 16) 5-TTTAATTCATGAGACGTCTTTGCGATCGCGCAGAAGAGAGAGAGAGAGAG GTAG- 3′.

The forward primer incorporates Avr II, PacI and PmeI restriction sitesand the reverse primer incorporates BspHI, Aat II and AsiSI andrestriction sites to facilitate further cloning steps. The resulting 1.1kb fragment (corresponding to nucleotides 101336-100216 of AP005570) iscloned into pCR®2.1-TOPO® vector using a TOPO TA Cloning® Kit(Invitrogen) and One Shot E. coli cells following manufacturer'sinstructions. The resulting plasmid is digested with restriction enzymesAvr II and BspH1 and is cloned into Nos/pCR2.1mut that has been digestedwith Avr II and Nco1. The resulting construct has an OsAnt1 promoter anda nos3′-region with unique AsiSI and AatII sites between them forcloning genes of interest. The expression cassette is flanked by Avr II,Pac I, and Pme I restriction sites on the 5′-end and a PacI restrictionsite on the 3′-end to facilitate movement into a plant binary expressionvector.

An expression cassette utilizing a sorghum Ant promoter is designed in asimilar manner. Forward primer 5′-ATTAAACCTAGGTTAATTAAGTTTAAACGATTCGACAATATTTATCAAAT-3′ (SEQ ID NO: 17) and reverse primer 5-TTTAATTCATGAGACGTCTTTGCGATCGCGGCGCCGGCGGC GTTGGCAGGT-3′ (SEQ ID NO: 18) can be used toamplify a 443-bp Ant promoter (SEQ ID NO:9) from sorghum genomic DNA asdescribed above for the OsAnt1 promoter and rice DNA. The clonedpromoter fragment is flanked by AvrII, Pac 1 and Pme 1 restriction siteson the 5′-end and BspHI, Aat II and Asi SI sites on the 3′-end. Thepromoter fragment is digested with restriction enzymes Avr II and BspH1and is cloned into Nos/pCR2.1mut that has been digested with Avr II andNco1. The resulting construct has a sorghum Ant promoter and anos3′-region with unique AsiSI and Aat II sites between them for cloninggenes of interest. The expression cassette is flanked by Avr II, Pac I,and Pme I restriction sites on the 5′-end and a Pad restriction site onthe 3′-end to facilitate movement into a plant binary expression vector.

An expression cassette utilizing a maize Ant promoter (see Example 5) isalso designed in a similar manner to that described for the rice andsorghum. Promoter regions from other antiquitin genes can also be usedas they are identified from genome sequencing projects and othertechnologies.

Example 7 Identification and Cloning of Alternate AlanineAminotransferase (AlaAt) Genes for Use in NUE Constructs

Aminotransferases are enzymes which catalyze the reversible transfer ofamino groups from amino acids to oxo acids. They can be divided intofour subgroups based on mutual structural relatedness (Mehta et al.,Eur. J. Biochem. 214(2):549-561, 1993). AlaAT enzymes catalyze thereversible interconversion of alanine and 2-oxoglutarate to pyruvate andglutamate and belong to subgroup 1. In addition to the barley alanineaminotransferase, other alanine aminotransferases are useful forconferring NUE in monocots.

To identify homologous AlaAT genes, the barley AlaAT protein sequence(NCBI accession number CAA81231) was used as a query to search the NCBIprotein sequence database using the BLAST algorithm. Genes with a highdegree of sequence homology to barley AlaAT were found in all majorclasses of eukaryotes. Related sequences were also found in bacteria. AtBlastn search of the NCBI EST database revealed that AlaAT homologs arewidespread in plants, but because most of these sequences were not fulllength they were not analyzed further. As additional genomic sequencesfor monocots become available, additional homologs may be identifiedusing these methods.

Full length sequences identified in the BLAST search were furtheranalyzed using the AlignX program (part of Vector NTI program suite,Invitrogen). A lineup of representative sequences and the correspondinghomology table using sequences from a range of organisms is shown inFIG. 2 and Table 1. The most homologous sequences were plant sequences.A lineup of representative plant sequences and the correspondinghomology table is shown in FIG. 3 and Table 2. Note that some ofsequences used for these alignments have been truncated so that theycontain less than the complete sequence of the cited AlaAT. Thealignment was performed using the methionine (M) of the barley AlaATsequence as the reference first residue.

mRNA Isolation and cDNA Synthesis

Tissue for RNA isolation was prepared from maize (A188) and rice(Nipponbare) in the following manner. Seeds were germinated in H₂O⁻ ongermination paper at 24° C. in a sealed bag (maize, rice). After 7 daysroot tissue was collected and stored in RNAlater® (Ambion) for RNAisolation. Seedlings of pepper (Capsicum annuum, Pepper Hot Asia,Santaka, Botanical Interests Broomfield, Colo.) were sterilized andgerminated in half strength MS and whole seedlings were used. Leavesfrom soil-grown Arabidopsis plants (Columbia 0) were used.

RNA was prepared from the plant tissues using the RNAqueous™-4PCR kit(Ambion). cDNA was synthesized from purified RNA using the SuperscriptIII Platinum® 2-step q-RT-PCR kit (Invitrogen) as per the manufacturer'sinstructions.

PCR Amplification of AlaAT

AlaAT genes may be amplified by PCR from cDNA from many sourcesincluding maize (Zea mays), rice (Oryza sativa), Arabidopsis thaliana,or pepper (Capsicum annuum). The template for barley (Hordeum vulgare L.cv Himalaya) AlaAT is plasmid pAG001 (obtained from Allen Good,University of Alberta) which contains the barley AlaAT coding sequencesas described in Muench and Good, 1994, GenBank accession CAA81231. PCRprimers contain an AsiS I restriction site on the 5′-end and an Aat IIrestriction site at the 3′-end to facilitate cloning into expressioncassettes. The primer pairs for the individual genes are listed below:

Barley Fw: (SEQ ID NO: 19)5′-ATTAAAGCGATCGCACCATGGCTGCCACCGTCGCCGTGGA-3′ Barley Rv:(SEQ ID NO: 20) 5′-TAGTGAGACGTCTTAGTCACGATACTCTGACA-3′ Maize Fw:(SEQ ID NO: 21) 5′-ATTAAAGCGATCGCACCatggccgccagcgtcaccgtgga-3′ Maize Rv:(SEQ ID NO: 22) 5-TAGTGAGACGTCTTAGTCGCGGTACTCGGCCAA-3′ Rice Fw:(SEQ ID NO: 23) 5′-ATTAAAGCGATCGCACCATGGCTGCTCCCAGCGTCGCCGT-3′ Rice Rv:(SEQ ID NO: 24) 5′-TAGTGAGACGTCTCAGTCGCGGTACGCTGCCATGAA-3′Arabidopsis At1gl7290 Fw: (SEQ ID NO: 25) 5′-ATTAAAGCGATCGCACCATGCGGAGATTCGTGATTGGCCAA-3′ Arabidopsis At1gl7290 Rv:(SEQ ID NO: 26) 5′- TAGTGAGACGTCTTAGTCGCGGAACTCGTCCATGAA-3′ Pepper Fw:(SEQ ID NO: 27) 5′-ATTAAAGCGATCGCACCATGGATTCCATCACTATTGAT-3′ Pepper Rv:(SEQ ID NO: 28) 5′-TAGTGAGACGTCTTAGCCGCAGAATTCATCCAT-3′

AlaAT genes may be amplified using the BD Advantage™ 2 PCR kit followingmanufacturer's instructions (Clontech, Mountain View, Calif.). Theresulting PCR products may be purified using QIAquick™ Purification Kit(Qiagen®, Hilden, Germany) and digested with AsiSI and Aat IIrestriction enzymes. The products may be ligated to the OsAnt1, sorghumAnt or maize Ant expression cassettes described above that have beendigested with AsiSI and Aat II restriction enzymes.

The AlaAT gene in each of the expression constructs is sequence verifiedfor PCR fidelity and integrity of the ATG start codon.

Example 8 Binary Vector Construction and Plant Transformation

The Ant promoter/AlaAT gene/nos 3′ expression cassettes are cloned intoa binary vector for plant transformation by digestion with Pme1 and Pac1and ligation with pARC110 digested with the same enzymes. pARC110 is anAgrobacterium binary vector originally based on pZP100 (Hajdukiewicz etal., Plant Mol. Biol. 25, 989-994, 1994). pARC110 utilizes a Bastaselectable marker driven by a CaMV 35S promoter and a nos terminator.The selectable marker is located near the left border, and the uniquerestriction sites Xba I, Avr II, Pac 1, and Pst I have been engineeredclose to the RB for gene cloning. The chloramphenicol bacterialselectable marker in the backbone of pZP100 was also replaced with thekanamycin resistance gene (nptIII) from the pCAMBIA 1304 vector (foundon the internet at the cambia.org website.

The promoter/AlaAT/nos 3′ gene binary vectors can be introduced intoAgrobacterium tumefaciens strains for Agrobacterium-mediatedtransformation of monocot crop plants or vector DNA is used for particlegun bombardment methods of plant transformation.

Example 9 Use of Alternate Antiquitin/AlaAt Constructs in RiceTransformation Using Selection on Bialophos

Agrobacterium-mediated rice transformation with the OsAnt1/AlaATconstruct, or any alternate Ant/AlaAT construct, is achieved using atransformation method based on the method described in U.S. Pat. No.7,060,876 and European Patent No. 672752B1. A detailed descriptionfollows.

Plasmids were transferred into Agrobacterium strain EHA105 (Hood et al.,Transgenic Res. 2: 208-218, 1993) by electroporation (Sambrook et al. inMolecular Cloning, A Laboratory Manual Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press, 1989). Agrobacterium cells were platedon solid AB medium (Chilton et al., 1974) containing 50 mg/l kanamycinand incubated at 28° C. for 3 days. The bacteria were then collectedwith a flat spatula and resuspended in liquid co-cultivation medium(R2-CL, Table 4) by gentle vortexing prior to transforming the ricetissues.

Mature seeds of rice (Oryza sativa L. cv. Nipponbare) were used in thetransformation experiment. The seeds were dehusked and surfacesterilized by dipping (1 min) in 70% (v/v) ethanol followed by soakingin 50% bleach plus 0.1% Tween-20 for 10 min and then rinsing five timesin sterile distilled water. Following sterilization, seeds were culturedon callus induction medium (N6C, Table 4) and incubated for three weeksin the dark at 26° C.

TABLE 4 Medium used for callus induction, inoculation, co-culture,resting phase, selection, regeneration and rooting Medium CompositionN6C N6 major salt, iron source, minor salts and vitamins Callusinduction medium (Chu (1975) Sci. Sin. 5: 659-668) + 3AA (100 mg/l(autoclave) myo-inositol + 500 mg/l L-proline + 500 mg/l L- glutamine) +300 mg/l casein hydrolysate + 2.0 mg/l 2,4-D + 30 g/l sucrose, pH 5.8,0.35% gellan gum R2-CL R2 major and minor salts, vitamins and ironsource Liquid co-culture medium without sucrose (Ohira et al. (1973)Plant and Cell (filter sterilize) Physiol. 14: 1113-1121) + 0.25Mglucose + 125 μM acetosyringone + 2.0 mg/l 2,4-D, pH 5.2 R2-CS R2-CL +0.35% gellan gum Solid co-culture medium (filter sterilize) N6S N6Cmedium + 200 mg/l Timentin + 7.5 mg/l Selection medium bialaphos, pH 5.8(filter sterilize) RN MS medium (Murashige & Skoog (1962) Physiol Plant15: Regeneration medium 473-497) + 2 mg/l kinetin + 0.02 mg/l NAA + 200mg/l Timentin + 7.5 mg/l bialaphos, pH 5.8, 0.35% gellan gum. R ½strength MS medium (Murashige & Skoog (1962) Rooting medium Physiol.Plant 15: 473-497) + 100 mg/l Timentin, pH 5.8, 0.35% gellan gum

After three weeks, 3-5 mm long embryogenic nodular units released fromthe scutellum-derived callus at the explant/medium interface wereimmersed into 25 ml of liquid co-culture medium (R2-CL, Table 4)containing Agrobacterium cells at the density of 10⁹ cells/ml(OD₆₀₀=0.3) in a 100 mm-diameter Petri dish for 10-15 minutes.Embryogenic units were then blotted dry on sterilized filter paper,transferred to a Petri dish containing solid co-culture medium (R2-CS,Table 4) and incubated for three days at 25° C. in the dark.Co-cultivated embryogenic calli were then transferred to N6 liquidmedium containing 400 mg/l Timentin for disinfection and placed for 4hours on an orbital shaker (100 rpm) at 26° C. in the dark. After dryblotting on sterile filter paper, calli were placed on N6 selectionmedium (N6S, Table 4) and kept at 26° C. in dark.

After 4 weeks of culture, uncontaminated embryogenic units had developedinto large yellowish globular structures that were transferred ontofresh N6S medium and cultured for another 4-5 weeks at 26° C. in dark.

The globular structures had proliferated many round-shaped, compact andyellowish calli. These putatively transgenic, bialaphos-resistant calliwere gently picked out, transferred and cultured on regeneration medium(RN, Table 4), incubated for 1 week in the dark, then maintained for 4-5weeks under a 14/10 hours day/night photoperiod with light provided atan intensity of 70 μmol/m per sec. Green shoots regenerating from aresistant callus were dissected and sub-cultured in culture vesselscontaining rooting medium (R, Table 4) for 2 weeks to promote vigorousroots and tillers before being transferred to 2-inch pots filled withsterile Sunshine Mix #3. The transgenic plantlets were acclimated bymaintaining them in growth rooms set to 26° C., 14/10 hours day/nightphotoperiod and high humidity. Fertilizer was applied three times a weekstarting two weeks after planting in pots. The fertilizer mix is SimmonsSolution (San Joaquin Sulphur Co., Lodi, Calif.) with addition ofcalcium nitrate. Sixteen g of Simmons and 60 g of calcium nitrate aremixed for 40 gallons of fertilizer.

Nitrogen efficient monocot plants including but not limited to maize,sorghum, barley, wheat, rye and grass can be developed using the methodsoutlined in the above examples.

The invention has been described with regard to one or more embodiments.However, it will be apparent to persons skilled in the art that a numberof variations and modifications can be made without departing from thescope of the invention as defined in the claims. The followingstatements of the invention are intended to characterize possibleelements of the invention according to the foregoing description givenin the specification. Because this application is a provisionalapplication, these statements may be changed upon preparation and filingof the complete application. Such changes are not intended to affect thescope of equivalents according to the claims issuing from the completeapplication, if such changes occur.

All citations are hereby incorporated by reference.

We claim:
 1. A transgenic monocot plant comprising a rice antiquitinpromoter operably linked with a nucleic acid encoding an alanineaminotransferase, wherein the rice antiquitin promoter comprises SEQ IDNO:1 or a sequence having 99.9% sequence identity to SEQ ID NO:1.
 2. Thetransgenic monocot plant of claim 1, wherein expression of said nucleicacid in said monocot plant causes an increase in plant dry weightbiomass as compared to the plant dry weight biomass of a comparablemonocot plant not expressing said nucleic acid when the plant expressingthe nucleic acid and the plant not expressing the nucleic acid are grownunder conditions that are limiting nitrogen conditions for the plant notexpressing the nucleic acid.
 3. The transgenic monocot plant of claim 2,wherein said alanine aminotransferase is selected from the groupconsisting of barley, rice, and maize, alanine amino transferases. 4.The transgenic monocot plant of claim 2, wherein the transgenic monocotplant is selected from the group consisting of barley, rice, sugar cane,maize, sorghum, rye, wheat and turfgrass.
 5. The transgenic monocotplant of claim 2, wherein the transgenic monocot plant is rice.
 6. Thetransgenic monocot plant of claim 2, wherein the rice antiquitinpromoter comprises SEQ ID NO:
 1. 7. The transgenic monocot plant ofclaim 2, wherein the rice antiquitin promoter comprises a sequencehaving 99.9% sequence identity to SEQ ID NO:1.
 8. The transgenic monocotplant of claim 1, wherein expression of said nucleic acid in saidmonocot plant causes an increase in seed weight as compared to the seedweight of a comparable monocot plant not expressing said nucleic acidwhen the plant expressing the nucleic acid and the plant not expressingthe nucleic acid are grown under conditions that are limiting nitrogenconditions for the plant not expressing the nucleic acid.
 9. Thetransgenic monocot plant of claim 8, wherein said alanineaminotransferase is selected from the group consisting of barley, rice,and maize, alanine amino transferases.
 10. The transgenic monocot plantof claim 8, wherein the transgenic monocot plant is selected from thegroup consisting of barley, rice, sugar cane, maize, sorghum, rye, wheatand turfgrass.
 11. The transgenic monocot plant of claim 8, wherein thetransgenic monocot plant is rice.
 12. The transgenic monocot plant ofclaim 8, wherein the rice antiquitin promoter comprises SEQ ID NO: 1.13. The transgenic monocot plant of claim 8, wherein the rice antiquitinpromoter comprises a sequence having 99.9% sequence identity to SEQ IDNO:1.
 14. Seed from the transgenic monocot plant of claim 1, wherein theseed comprises the rice antiquitin promoter operably linked with thenucleic acid encoding the alanine aminotransferase.
 15. The transgenicmonocot plant of claim 2, wherein said alanine aminotransferase isselected from the group consisting of Hordeum vulgare, Panicummiliaceum, Oryza sativa, Zea mays, Arabidopsis thaliana, and Capsicumsp. alanine amino transferases.
 16. The transgenic monocot plant ofclaim 8, wherein said alanine aminotransferase is selected from thegroup consisting of Hordeum vulgare, Panicum miliaceum, Oryza sativa,Zea mays, Arabidopsis thaliana, and Capsicum sp. alanine aminotransferases.